Our long-term objective is to elucidate basic mechanisms regulating intestinal mucosal functions, with particular emphasis on epithelial cell proliferation and differentiation, and their modulation by growth factors and hormones. The proposed research will utilize a new in vitro model we have developed, centered on human intestinal epithelial cells conditionally-immortalized with a temperature-sensitive SV40 large tumor antigen (tsFHI cell lines). These cells can be maintained without apparent limits in a proliferative state by culture at 32 degrees C, and induced to differentiate at will by a shift to a higher temperature at which the oncogene is inactivated. Using these cells, we will investigate the role and mechanism of action of recently discovered cell cycle regulators, the cyclin-dependent kinase inhibitors (CKIs), and other yet to be identified cellular components, in the intestinal cell differentiation process. Our specific aims are: 1) To obtain direct evidence for a key role of the CKI p21/Waf-1/Cip1 in the irreversible loss of proliferative potential that marks the onset of tsFHI cell differentiation; 2) To correlate changes in expression and complex formation of other known CKIs with tsFHI cell growth and differentiation; and 3) To identify, clone, and characterize other cellular components whose expression is specifically altered at the time tsFHI cells are induced to differentiate. The methods that will be used in these studies will include immunoprecipitation, Western and Northern blotting, transfection with wild-type or deleted cDNAs, mRNA differential display by PCR, cDNA cloning, sequencing, and analysis of function of cloned genes. The proposed studies should provide information important to further our understanding of a variety of intestinal physiological activities and disease states, in particular nutrition and carcinogenesis.